Compositions for the prevention and treatment of viral-induced tumors

ABSTRACT

Prophylactic and therapeutic agents for the prevention and treatment of viral-induced tumors and, in particular, compositions derived from an Aspergillus fermentation extract, and the use thereof, either directly or to prepare a medicament, for the prevention and treatment of viral-induced tumors in mammals. Such tumors include papilloma-induced tumors. The composition is topically administered.

This is a Continuation-in-Part of application Ser. No. 08/126,660, filedSep. 27, 1993 now U.S. Pat. No. 5,500,359.

FIELD OF THE INVENTION

The present invention relates to prophylactic and therapeutic agents forthe prevention and treatment of viral-induced tumors and, in particular,to compositions derived from Aspergillus fermentation extracts for useas a topical agent for the prevention and treatment of viral-inducedtumors, such as papillomavirus-induced tumors, in mammals.

BACKGROUND OF THE INVENTION

Viruses which induce tumors in mammals are quite widespread. Indeed,there are over sixty-eight types of human papillomaviruses (HPV) alonewhich can induce the production of tumors. Some of these HPV's have beenassociated with benign tumors, such as common warts, while others havebeen strongly implicated as etiologic agents in dysplasia and carcinomasin the oral and genital mucosa of the infected mammal. Other types ofviruses which can result in tumors include various RNA viruses as wellas herpes viruses.

Recently, it has also been observed that individuals with depressedimmune systems, such as sufferers of Aquired Immune Deficiency Syndrome(AIDS), are prone to human papillomavirus infections which can result intumor growth over their entire bodies, resulting in great mental andphysical distress to the afflicted individual.

Current modalities for the treatment of viral-induced tumors involve theremoval of the tumor by either: (1) surgical intervention (laser oroperative); (2) the application of organic acids, such as glacial aceticacid and/or salicyclic acid, to "burn" the tumor away; (3) the injectioninto the tumor of an anti-tumor vaccine prepared from ground tumors;and, to a lesser extent, (4) the application of a drug treatment [suchas podophyllin; interferons and 5-fluoro-2,4 (1H,3H)-pyrimidine-dione;2-4-dioxo-5-fluoropyrimidine--also referred to asfluorouracil or 5-FU].

While being useful for removing the viral-induced tumor, the currenttreatment modalities presently used nonetheless suffer from one or moreof the following drawbacks: (1) they can result in the destruction ofhealthy uninfected tissue; (2) they can result in scarring anddisfigurement; (3) they can result in discomfort to the mammal beingtreated thereby; and (4) they do not always result in the destruction oflatent viral DNA which may be maintained in surrounding tissues.Furthermore, with these treatments, patients have suffered fromsignificant systemic side effects, incomplete resolution and frequentrecurrences of the tumors.

It has also been disclosed to use phototherapy for removing laryngealpapillomatosis tumors. While such phototherapy reduced tumor growth byabout 50%, it also resulted in a generalized skin photosensitivity forat least six weeks, as well as other minor reactions. Furthermore,despite the apparent success of this technique, the presence of latentviral DNA is nonetheless still maintained in the surrounding tissues.

U.S. Pat. No. 5,073,630 discloses a polymeric anhydride of magnesium andproteic ammonium phospholinoleate with antiviral, antineoplastic andimmunostimulant properties. This antiviral agent was produced in thecell-free filtrate of a selected line of Aspergillus sp. However, thatcompound is insoluble in water and possesses a high molecular weight(316,000 daltons). Further, recovery of that compound presents problems.

Accordingly, it can be seen that there remains a need for prophylacticand therapeutic compositions capable of preventing and treatingviral-induced tumors in mammals without either destroying healthyuninfected tissue, causing significant systemic side effects, causingscarring or disfigurement of, and/or discomfort to, the mammal treatedtherewith, and which results in the destruction of latent viral DNAwhich may be maintained in surrounding tissues, so that instances ofincomplete resolution and frequent recurrences of the tumors arereduced. It can further be seen that there also remains a need formethods for providing such prophylactic and therapeutic compositions, aswell as methods for the use of such prophylactic and therapeuticcompositions for the prevention and treatment of viral-induced tumors inmammals.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide prophylacticand therapeutic compositions for the prevention and treatment ofviral-induced tumors in mammals which compositions neither destroyhealthy uninfected tissue, nor result in either significant systemicside effects, scarring, disfigurement or discomfort to the mammaltreated therewith, and further which result in the destruction of latentviral DNA which may be maintained in surrounding tissues, so thatinstances of incomplete resolution and frequent tumor recurrence arereduced.

It is a further primary object of the present invention to providesimple, easy to perform methods for providing such prophylactic andtherapeutic compositions for the prevention and treatment ofviral-induced tumors in mammals.

It is a still further primary object of the present invention to providemethods for preventing and treating viral-induced tumors in mammalswhich methods neither destroy healthy uninfected tissue, nor result ineither significant systemic side effects, scarring, disfigurement ordiscomfort to the mammal treated therewith, and further which result inthe destruction of latent viral DNA which may be maintained insurrounding tissues, so that instances of incomplete resolution andfrequent tumor recurrence are reduced.

In accordance with the teachings of the present invention, disclosedherein are prophylactic and therapeutic compositions for the preventionand treatment of viral-induced tumors in mammals. These compositionsneither destroy healthy uninfected tissue, nor result in eithersignificant systemic side effects, scarring, disfigurement or discomfortto the mammal treated therewith. Furthermore, these compositions resultin the destruction of latent viral DNA maintained in surroundingtissues, so that instances of incomplete resolution and frequent tumorrecurrence are reduced.

It is preferred that these prophylactic and therapeutic compositions besuitable for topical uses.

Preferably, the prophylactic and therapeutic compositions of the presentinvention are fermentation extracts and/or derivatives thereof. It isfurther preferred that these fermentation extracts be an Aspergillusfermentation extract. A preferred Aspergillus fermentation extract is anAspergillus niger fermentation extract. Especially preferred areAspergillus niger 1.2 AN29 and Aspergillus niger 1.2 AN39 fermentationextracts.

It is still further preferred that the compositions of the presentinvention include these fermentation extracts and/or derivatives thereofin a pharmaceutically-acceptable carrier.

In a preferred embodiment, the compositions of the present inventioninclude concentrated fermentation extracts. If desired, these extractsmay be freeze-dried.

In a particularly preferred embodiment, these fermentation extracts arecrude cell-free whole extracts. More particularly, such crude cell-freewhole extracts are ultrafiltered through a 10000 molecular weight (MW)cut-off membrane, whereby the retentate is a 10000 molecular weight (MW)permeate.

If desired, the compositions of the present invention may be enzymecompositions which are either derivatives of and/or have been derivedfrom the fermentation extract(s) disclosed herein.

In a particular aspect of the present invention, the Aspergillusfermentation extracts (and/or derivatives thereof) described herein areused for the preparation of prophylactic and therapeutic compositionsfor the prevention and treatment of viral-induced tumors in mammals.Preferably, the Aspergillus fermentation extracts (and/or derivativesthereof) are used for the preparation of such prophylactic andtherapeutic compositions which may be topically applied to a mammal inneed thereof.

In further accordance with the teachings of the present invention,disclosed herein are methods for providing the prophylactic andtherapeutic compositions of the present invention. These methods includeculturing an Aspergillus species on a suitable medium. Preferably, thismedium is a solid surface medium. These methods further includeextracting extracellular compounds from the medium with water, so that aliquid fermentation extract is obtained. These methods further includefiltering the obtained fermentation extract to remove cell biomass andspores therefrom, thereby providing a liquid fermentation extract.Finally, and if desired, the disclosed methods include refrigerating theliquid fermentation extract until the use thereof. Preferably, suchrefrigeration is done at about 4° C.

In another particularly preferred embodiment, the fermentation extractmay be concentrated by freeze-drying. The freeze-dried powder (of thefermentation extract) that results from such freeze-drying may be storedat room temperature with a dessicant until use.

In a particular preferred embodiment, the fermentation extract isfurther ultrafiltered through a 10000 MW cut-off membrane before theretentate is refrigerated. This 10000 MW ultrafilter retentate has a drysolids content of 7.6% (w/v). If desired, the retentate may then beconcentrated by, for example, evaporation.

Preferably, the methods disclosed herein are used for the preparation oftopical prophylactic and therapeutic compositions.

In yet further accordance with the teachings of the present invention,disclosed herein are methods for the prevention and treatment ofviral-induced tumors in mammals. Use of these methods neither destroyhealthy uninfected tissue, nor result in either significant systemicside effects, scarring, disfigurement or discomfort to the mammaltreated therewith. Furthermore, use of these methods results in thedestruction of latent viral DNA maintained in surrounding tissues, sothat instances of incomplete resolution and frequent tumor recurrenceare reduced. These methods include preparing a fermentation extractand/or a derivative thereof in a pharmaceutically-acceptable carrier, sothat a prophylactic and/or therapeutic composition is provided for theprevention and/or treatment of viral-induced tumors in mammals. Thesemethods further include administering a therapeutically-effective amountof the composition to a mammal in need thereof. Such administration may,in the case of a prophylactic treatment, be on that area of the mammalon which it is anticipated that such preventive treatment is needed or,in the case of a therapeutic treatment, be directly on the viral-inducedtumor of the mammal in need thereof. Preferably, such application is atopically performed.

Preferably, providing the prophylactic and therapeutic compositions ofthe present invention includes preparing a fermentation extract, and inparticular a fermentation extract of Aspergillus. More preferably, themethod involves providing a fermentation extract of Aspergillus niger.Most preferably, this method involves providing a fermentation extractof Aspergillus niger 1.2 AN29 or 1.2 AN39.

If desired, the method may further include the preparation of aderivative of the fermentation extract.

If desired, the method may further include ultrafiltering the cell-freewhole A. niger fermentation extract through a 10000 MW cut-off membraneand recovering of the 10000 MW ultrafilter retentate. If furtherdesired, the method may further include concentrating the retentate byevaporation.

If desired, the method may further include freeze-drying thefermentation extract, wherely the extract is concentrated and a powderformed, storing of the freeze-dried powder may be done at roomtemperature en the presence of a dessicant.

In a particular aspect of the present invention, disclosed herein areprophylactic and therapeutic topical compositions and methods for theuse thereof for the prevention and treatment of Epstein-BarrVirus-induced tumors in mammals.

In a further particular aspect of the present invention, disclosedherein are prophylactic and therapeutic topical compositions and methodsfor the use thereof for the prevention and treatment of cottontailrabbit papillomavirus-induced tumors in mammals.

In still another particular aspect of the present invention, disclosedherein are prophylactic and therapeutic topical compositions and methodsfor the use thereof for the prevention and treatment ofpapillomavirus-induced tumors in mammals.

These and further objects and advantages of the present invention willbecome readily apparent upon a reading of the following invention inconjunction with the examples thereof.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention includes prophylactic and therapeutic topicalcompositions for the prevention and treatment of viral-induced tumors inmammals. These compositions are prepared from fermentation extractsand/or derivatives thereof. These fermentation extracts and derivativesthereof may then be mixed with a pharmaceutically-acceptable carrier tomanufacture the therapeutic compositions of the present invention.

By the term "fermentation extracts" what is referred to are extracts ofthe milieu, and in particular the fermentation milieu, which has beeninoculated with a culture of a suitable microorganism and in which themicroorganism has been cultured (grown).

By the terms "derivatives of" and "derived from" when used in referenceto the fermentation extracts, what is meant are compositions orcomponents, such as particular enzymes or combinations of enzymes, whichhave either been obtained (derived or isolated) from the fermentationextracts. By way of illustration, an example of such a derivative wouldbe a fermentation extract which has been concentrated or filtered. Theterms "derivatives of" and "derived from" are used equally to refer tocompositions and compounds which are both identical to thosecompositions or components of the fermentation extract and whichdemonstrate the same prophylactic and therapeutic properties of thefermentation extracts. By way of illustration, such definition includescomponents of the fermentation extract itself (such as the active agentthereof) which have been isolated (and, if desired, purified) from thefermentation extract. By way of further illustration, such definitionalso includes compositions or compounds which have been constructed(i.e., synthetically constructed) to mimic the active agent(s) havingthe prophylactic and therapeutic properties of the fermentation extractsof the present invention.

More particularly, the fermentation extracts and derivatives thereofwhich are disclosed herein are water extracts of surface fermentations(or derivatives of such water extracts) which have been inoculated witha culture of a suitable microorganism.

The fermentation extracts are, preferably Aspergillus fermentationextracts and, more particularly, an Aspergillus niger fermentationextracts. Most preferred are Aspergillus niger 1.2 AN29 fermentationextracts and Aspergillus niger 1.2 AN39 fermentation extracts.

In a particular preferred embodiment, the fermentation extracts are10000 MW ultrafiltered retentates (having a dry solids content of 7.6%(w/v). The retentate may be concentrated by, for example, evaporation.

In particular, it has been observed that water extracts of surfacefermentations inoculated with an Aspergillus niger culture contributesto the prevention of and the disappearance of warts in mammals.

The pharmaceutically-acceptable carrier may be any such carrierwell-known to those skilled in the art. It is preferred that such acarrier be a hydrophilic substance that aids the prophylactic andtherapeutic compositions to penetrate the skin. Examples of suchcarriers include, but are not limited to, aqueous menthol solutions,water, propylene glycol, lanolin, butyl alcohol, absolute alcohol,isopropyl alcohol, dimethyl sulfoxide, ether ethyl lactate and mixturesthereof. Another example of such a carrier is the well-known "VEHICLEN", a composition comprised of ethyl alcohol, isopropyl alcohol,purified water, LAURETH-4 (a surfactant) and propylene glycol.

The precise amounts of the fermentation extract (and/or derivativesthereof) and the pharmaceutically-acceptable carrier, to employ inpreparing the prophylactic and therapeutic compositions of the presentinvention are, preferably, a concentration of about 1% (w/v) to about15% (w/v) of freeze-dried fermentation extract (or derivative thereof).A concentration of about 8% (w/v) of freeze-dried fermentation extractis especially preferred.

The prophylactic and therapeutic compositions may be formulated asdesired for topical application on either the area of the mammal to beprotected or on the afflicted tumor, such as a wart, of the mammal inneed thereof. Such formulations include liquid compositions, such asoil-based ointments, linaments and tincture compositions. Creams, soapsand gels are especially preferred for their ability to keep theprophylactic and therapeutic compositions in prolonged contact with theskin and/or tumor for a sufficient period of time.

While not precisely understood, it is believed that the mode of actionof the compositions of the present invention may not necessarily bedirectly antiviral or antitumor in nature. Rather, it is believed thatis it possible that the mode of action of the compositions disclosedherein may be the result of a general stimulation of the immune system.Cell-mediated responses may be important for the AN-1 antigen which mayinduce a generalized immunological response which affects tumor growth.In this respect, we believe that the compositions of the presentinvention may be immunopotentiators. Indeed, previous studies have shownthat a cell-mediated response is most likely responsible for regressionof papillomavirus-induced for benign and pre-malignant neoplasia.

It is also believed that the active component of the fermentationextract has a molecular weight of between about 1000 daltons and about10000 daltons. More particularly, it is believed that the activecomponent of the fermentation extract has a molecular weight ofapproximately 10000 daltons.

The prophylactic and therapeutic compositions of the present inventionare useful for the prevention and treatment of viral-induced tumors,such as those resulting from human papilloma viruses (HPV), cottontailrabbit papilloma virus (CRPV), equine papillomavirus (EPV) and bovinepapillomavirus (BPV).

The prophylactic and therapeutic compositions of the present inventionare provided by, first, preparing a suitable growth medium. Preferred isa solid surface medium. The precise medium to utilize will varyaccording to the microorganism to be cultured thereon, as is well withinthe skill of the art to ascertain. In the event that an Aspergillus isto be cultured thereon, it is preferred that the medium includes: 7.9%(w/w) SOLKA FLOC BNB 100 (James River Corp., U.S.A.); 7.9% (w/w) oathulls; 7.9% (w/w) peanut meal; 15.8% (w/w) beet pulp; 0.39% (w/w) KH₂PO₄ ; 13 ppm ZnSO₄ ; and 60% (w/w) water.

The medium is then sterilized, cooled and inoculated with spores of theprecise Aspergillus species to be cultured thereon. After inoculationand mixing, the medium is transferred to porous metal trays at a depthof approximately 0.75 inches. These trays are then incubated in a highhumidity environment at 30° C. to 32° C. for about 72 hours during whichtime the Aspergillus species is cultured. The contents are thenharvested (extracted) by stirring in water (water extraction) forseveral hours, so that a liquid fermentation extract is obtained. Theliquid fermentation extract is then subjected to a filtration (passedthrough a final filter) to remove cell biomass, spores and otherinsolubles from the extract.

If desired, the liquid filtered extract may then be used as such, or itmay be further processed, such as by being concentrated, so that asuitable derivative thereof is provided.

An example of such further processing includes ultrafiltration of thefermentation extract through a 10000 MW cut-off membrane, so as toobtain a 10000 MW-UF retentate. Such a retentate may, for example, havea dry solids composition of 7.6% (w/v). Another example of such furtherprocessing is concentration (i.e., 2.5 fold) by evaporation. In thisregard, it is noted that either the crude cell-free fermentation extractor the 10000 MW (or any other size) retentate may be so concentrated byevaporation.

If not to be used immediately, the liquid filtered extract (and/orderivative thereof) is, preferably, refrigerated at 4° C. until use.

If long-term storage is desired, the liquid filtered extract (orderivative thereof) may then be vacuum concentrated, followed by aclarifying filtration. The filtrate (or derivative thereof) may then befreeze-dried. The freeze-dried powder resulting therefrom may then bestored at room temperature with a dessicant until use.

The resulting freeze-dried powder of the fermentation extract (orderivative thereof) is very soluble in water. When use thereof isdesired, the freeze-dried powder may be redissolved in a liquid, such aswater, and/or in the pharmaceutically-acceptable carrier.

The prophylactic and therapeutic compositions of the present inventionmay be used in methods for the prevention and treatment of viral-inducedtumors in mammals. These methods include administering atherapeutically-effective amount of the prophylactic and therapeuticcompositions including an Aspergillus fermentation extract (or aderivative thereof) in a pharmaceutically-acceptable carrier to a mammalin need thereof.

By the term "therapeutically-effective amount" what is meant is anamount which is effective for either prophylactic or therapeuticpurposes to prevent or mitigate the growth of new or existingviral-induced tumor(s) in question.

The precise amount of the prophylactic and therapeutic compositions tobe applied is that quantity of the topical prophylactic and therapeuticcompositions of the present invention which is necessary to thinlysaturate the (afflicted) area of the skin (such as that area where theviral-induced tumor is located) of a mammal in need thereof.

The prophylactic and therapeutic compositions can be administered in anysuitable manner well-known to those skilled in the art. Such methods caninclude subcutaneous or intravenous injection. Preferably, thisadministration is a topical administration, such as by being applied tothe surface of the skin or tumor (or afflicted area in need thereof)with the aid of an eyedropper, a porous applicator (such as a gauze,swab or cloth, a roll-top applicator) a brush or any other suitableapplication means, as desired.

If desired, the prophylactic and therapeutic compositions may be appliedbefore infection in a prophylactic treatment to prevent initialinfection. We have found that the prophylactic and therapeuticcompositions have significant tumor reducing potential when appliedeither shortly after infection, or when applied to existing tumors. Itis contemplated that such applications will be performed at least one totwo times per day as long as the tumors persist. However, the precisefrequency of these applications may be increased and/or decreased asdesired or needed, as is well within the skill of the art to determine.Care should be taken to avoid the development of skin reactions to thecompositions, but in any event, we have found that any such reactionseventually diminished without any harm to the specimen being tested.

Having thus described the prophylactic and therapeutic compositions ofthe present invention, as well as the method for the preparation thereofand the use thereof, the following examples are now presented for thepurposes of illustration only and are neither meant to be, nor shouldthey be, read as being restrictive.

EXAMPLE 1 Preparation of an Aspergillus niger 1.2 AN39 FermentationExtract

First, a suitable growth medium was prepared comprised of 7.9% (w/w)SOLKA FLOC BNB 100 (James River Corp., U.S.A.); 7.9% (w/w) oat hulls;7.9% (w/w) peanut meal; 15.8% (w/w) beet pulp; 0.39% (w/w) KH₂ PO₄ ; 13ppm ZnSO₄ ; and 60% (w/w) water.

The medium was then sterilized, cooled and inoculated with spores ofAspergillus niger 1.2 AN39 which has been deposited under the provisionsof the Budapest Treaty in the Agricultural Research Service CultureCollection (NRRL), 1815 N. University St., Peoria, Ill. (U.S.A.) on Jul.30, 1993 under Accession Number 21126.

After inoculation and mixing, the medium was transferred to porous metaltrays at a depth of approximately 0.75 inches. These trays were thenincubated in a high humidity (water-saturated) environment at 30° C. to32° C. for about 72 hours to produce the desired Aspergillus niger 1.2AN39 cultures. The cultures were then harvested by stirring in water(water extraction) for several hours followed by filtration (passedthrough a final filter) to remove cell biomass, spores and otherinsolubles.

The filtrate was then vacuum concentrated, followed by a clarifyingfiltration. The resulting composition was an Aspergillus niger 1.2 AN39fermentation extract which was designated "AN-1".

A portion of the liquid filtered extract was then freeze-dried producingan AN-1 fermentation Extract powder. This freeze-dried powder of thefermentation extract was then stored at room temperature with adessicant until the use thereof.

EXAMPLE 2 Preparation of an Aspergillus niger 1.2 AN29 FermentationExtract

An Aspergillus niger 1.2 AN29 fermentation extract was prepared in thesame manner as the Aspergillus niger 1.2 AN39 fermentation extractdescribed above in Example 1, with the exception that the cooled,sterilized medium was inoculated with spores of Aspergillus niger 1.2AN29 which has been deposited under the provisions of the BudapestTreaty in the Agricultural Research Service Culture Collection (NRRL),1815 N. University St., Peoria, Ill. (U.S.A.) on Sep. 7, 1993 underAccession Number 21139. This composition was designated "AN-2".

A portion of the liquid filtered extract was then freeze-dried producingan AN-1 fermentation Extract powder. This freeze-dried powder of thefermentation extract was then stored at room temperature with adessicant until the use thereof.

EXAMPLE 3 In Vitro Therapeutic Index of A. niger Fermentation Extract

An estimation of the efficacy of the enzyme composition of the presentinvention against Epstein-Barr virus (EBV), a herpes-type virus, invitro, was determined to ascertain the potential usefulness of thetherapeutic composition of the present invention in vivo.

A standard estimation of the potency of antiviral agents is by acomparison of the ratio of the ED₅₀ obtained for cytotoxicity to theED₅₀ obtained for viral inhibition. This relationship is referred to asthe "in vitro therapeutic index" or "selectivity index". Relative to theinstant matter, the selectivity index of antiviral agents must be morethan 100 to indicate a useful effect on viral inhibition in animalexperiments as described in (1).

The estimation made herein was performed by a superinfection of Rajicells with a EBV viral cell line commonly referred to as P3HR-1. P3HR-1is a standard laboratory EBV strain. After superinfection, the cultureswere assayed for early antigen production.

1. Preparation of Human Foreskin Fibroblast Cells

Newborn human foreskins were obtained as soon as possible aftercircumcisions were performed and placed in Dulbecco's Minimal EssentialMedium (DMEM) (GIBCO BRL, Life Technologies, Inc., Gaithersburg, Md.U.S.A.) supplemented with (per ml DMEM) 50 μg vancomycin, 3 μgfungizone, 100 units penicillin and 25 μg gentamycin at 37° C. for 4hours.

The medium was then removed, the foreskin minced into small pieces andrepeatedly washed with Dulbecco's phosphate buffered saline (DPBS)(GIBCO BRL), from which the calcium and magnesium were ommitted, untilred cells were no longer visually present.

The tissue was then trypsinized using trypsin at 0.25% (w/v) withcontinuous stirring for 15 minutes at 37° C. in a CO₂ incubator. At theend of the 15 minute period, the tissue was allowed to settle to thebottom of the flask. The supernatant containing cells was then pouredthrough a sterile cheesecloth and into a second flask containing DMEMand 10% (v/v) fetal bovine serum (Hyclone, U.S.A.). After eachfiltration of cells, the cheesecloth was washed with a small amount ofDMEM-containing serum. Fresh trypsin was again added to the foreskinpieces as described above and the procedure was repeated until no morecells became available.

The second flask (containing the medium and the trypsinized cells) waskept on ice throughout the trypsinizing procedure.

The cell-containing medium in the second flask was then centrifuged atapproximately 1000 RPM (about 100 g) at 4° C. for ten minutes (theminimum centrifugal force required to pellet the cells without causingdamage thereto). The supernatant liquid was discarded and the cellsresuspended in about 50 ml of DMEM with 10% (v/v) fetal bovine serum.

The cells were then placed in an appropriate number of 25cm² tissueculture flasks. The cells were kept on 50 μg/ml DMEM vancomycin and 3μg/ml DMEM fungizone at 37° C. for about 72 hours until the cells weresubconfluent. The cells were then subcultured as described above, but inlarger flasks and with fresh medium. This procedure was then repeatedtwo more times until four passages were achieved.

2. Screening Assays for EBV

A. Virus

The prototype of infectious EBV used was the virus derived fromsupernatant fluids of the P3HR-1 cell line (obtained from the AmericanType Culture Collection (ATCC), Rockville, Md. (U.S.A.) under AccessionNumber VR603) following the procedure described in (12). This cell lineproduces nontransforming virus that causes the production of earlyantigen (EA) after primary infection or superinfection of B cell lines.

B. Cell Lines

Raji (obtained from the American Type Culture Collection (ATCC),Rockville, Md. (U.S.A.) under Accession Number CCL86) is a Burkitt'slymphoma cell line containing 60 EBV genomes/cell and was the primarycell used for screening antiviral activity against EBV early antigen(EA) expression.

All viral cell lines (the P3HR-1 as well as the Raji viral cell lines)were maintained in RPMI-1640 medium (GIBCO BRL) supplemented by 10%(v/v) fetal bovine serum, 2.05 mM/ml medium L-glutamine and 25 μg/mlmedium gentamycin. Twice weekly half of the medium volume was replacedwith fresh medium and the cell concentration adjusted to 3×10⁵ /ml asdescribed by (12). The cells were then maintained at 37° C. in anhumidified (90%) atmosphere with 5% (v/v) CO₂ until used.

3. Immunofluorescence Assays with Monoclonal Antibodies

The Raji cells were infected as described by (12) with the P3HR-1 strainof EBV. The composition, obtained as described above in Example 1, wasthen added after passive adsorption for 45 minutes at 37° C. and washingof the cell cultures with Dulbecco's phosphate buffered saline (DPBS)but without calcium and magnesium. The cultures were then incubated at37° C. for two days in RPMI-1640 medium (described above) to allow viralgene expression. Following the 48 hour incubation period, the number ofcells of each sample were counted with a hemacytometer as described in(12) and then spotted onto wells of Toxoplasmosis slides (Bellco GlassCo., U.S.A.) and air-dried.

Monoclonal antibodies (graciously provided by Dr. Gary Pearson,Georgetown University, U.S.A.) to the diffuse early antigen EA(D)(DuPont, U.S.A.) was then added to the Raji cells in the wells in theslides as described in (12). This was followed by the addition of afluorescein conjugated goat anti-mouse IgG antibody (Fisher Scientific,U.S.A.), following the procedure described in (12) and the number offluorescence positive cells in the wells of the slides were visuallycounted using a fluorescense microscope. The total number of cells inthe cultures positive for EA(D) were then calculated and compared asdescribed by (12), the early antigen expression of the EBV beinginhibited in those Raji cells that did not exhibit fluorescence.

4. In Vitro Cytotoxicity

In vitro cytotoxicity of AN-1 was determined in human foreskinfibroblast (HFF) cells, obtained as described above, following thetechnique and under the conditions described by (13) modified asfollows: twenty-four hours prior to assay, low passage HFF cells wereplated into 96-well tissue culture plates (having 8×12 flat-bottomwells) (Becton Dickinson Labware, U.S.A.) a concentration of 2.5×10⁴cells per well. The cells were in 0.1 ml of DMEM containing 10% (v/v)fetal bovine serum (Hyclone, U.S.A.). The cells were then incubated fortwenty-four hours at 37° C. in a CO₂ incubator. The medium was thenaspirated and 100 μl of DMEM containing 2% (v/v) of fetal bovine serumwas added to all but the eight wells in the first row. Freeze-driedfermentation extract (AN-1), obtained as described above in Example 1,was dissolved in DMEM containing 2% (v/v) fetal bovine serum to aconcentration of 100 μg/ml. 125 microliters of AN-1 was then added toeach respective well in the first row and the AN-1 was then diluted[with DMEM containing 2% (v/v) fetal bovine serum] serially 1:5 (givingan AN-1 concentration range in the wells of from 100 μg/ml to 0.03μg/ml) throughout the remaining wells by transferring 25 μl using theCetus Liquid Handling Machine (Perkin-Elmer Corp., U.S.A.). The plateswere then incubated for seven days in a CO₂ incubator at 37° C. Then,the cell-free medium containing AN-1 solution was aspirated and 200μl/well of 0.01% (v/v) neutral red in Dulbecco's Phosphate BufferedSolution was added. This was incubated at 37° C. in a CO₂ incubator forone hour. The dye was then aspirated and the cells were washed using aNunc Plate Washer (Nunc, Inc., U.S.A.). After removing the DPBS wash,200 μl/well of 50% (v/v) EtOH/1% (v/v) glacial acetic acid (in H₂ O) wasadded. The contents were mixed by rotating the plates on an orbitalshaker for 15 minutes. Then, the optical density of each well was readat 550 nm with a plate reader (Beckman Instruments Inc., U.S.A.).

Visual inspection of the HFF cells in each assay system (generallystationary cells), which were treated with 100 μg/ml of AN-1, indicatedno toxicity.

Also, the cytotoxicity of AN-1 has been determined in the HFF cellproliferation assay (rapidly-growing HFF cells). The cell proliferationassay of AN-1 for rapidly-growing human foreskin fibroblast cells wasdone. Twenty-four hours prior to assay, HFF cells (obtained as describedabove) were seeded in six-well tissue culture plates (having 2×3 flatbottom wells) (Becton Dickinson Labware, U.S.A.) at a concentration of2.5×10⁴ cells per well in DMEM containing 10% (v/v) fetal bovine serum.On the day of the assay, AN-1 was diluted serially in DMEM containing10% (v/v) fetal bovine serum at increments of 1:5 giving an AN-1concentration in each of the various wells covering a range of from 100μg/ml to 0.03 μg/ml. The medium from the cells was then aspirated and 2ml of AN-1 concentration was then added to each well. The cells werethen incubated in a CO₂ incubator at 37° C. for seventy-two hours. Then,the cell-free medium containing AN-1 in solution was removed and thecells (monolayer) washed with DPBS that contained neither calcium normagnesium. The DPBS was then removed by aspiration. One ml of 0.25%(w/v) trypsin was added to each well and incubated until the cellsstarted to become separated from the bottom of the wells of the plate.The cell-medium mixture was then pipetted up and down vigorously tobreak up the cell suspension and 0.2 ml of the mixture was added to 9.8ml of the diluent ISOTON III (Coulter Electronics Inc., U.S.A.) and thecells counted using a Coulter Counter (Coulter Electronics Inc.,U.S.A.). Each sample was then counted three times with three replicatewells per sample.

There was no toxicity of AN-1 at the 100 μg/ml level with this morestringent assay for toxicity.

Also, AN-1 was not toxic to the untransfected (EBV-free) Raji Burkitt'sLymphoma cell line at the 100 μg/ml level (IC₅₀ being greater than 100μg). The method that was used for the toxicity assay of AN-1 foruntransfected (EBV-free) Raji Burkitt's Lymphoma cells was described by(12) at page 85.

5. Results

The results of these screenings are given below, wherein: EC₅₀ (50%effective concentration) is the concentration required to inhibit viralcytopathogenicity by 50%; IC₅₀ (50% inhibitory concentration) is theconcentration required to inhibit cell proliferation by 50%; and S.I.stands for "Selective Index". The SI=IC₅₀ /EC₅₀. (1).

When the antiviral activity of AN-1 to early antigen (EA) expression ofEpstein-Barr virus (EBV) in RAJI cells (˜60 EBV copies/cell) was tested,the selectivity index (SI) obtained was greater than 137 (IC₅₀ >100μg/ml÷EC₅₀ 0.73 μg/ml), as follows:

EBV (RAJI Cells)

Immunofluoresence--Micrograms (MCG)/ml EC₅₀ =0.73; IC₅₀ >100; SI>137

Since the SI of AN-1 was more than 100 (>137) in RAJI cells, a usefuleffect on viral inhibition in mammal experiments is indicated.

The EC₅₀ for acyclovir (an antiviral standard which has inhibitoryactivity towards several herpes viruses) was 4.9 μg (ACV EC₅₀ 4.9).Thus, the crude AN-1 sample was 6.71 times more potent than acyclovir.It is anticipated that, after it is purified, the specific activity ofthe AN-1 composition would be increased.

[Acyclovir is2-Amino-1,9-dihydro-9-[(2-hydroxy-ethoxy)methyl]-6H-purin-6-one;acycloguanosine; 9-[(2-hydroxyethoxy)methyl]guanine. C₈ H₁₁ N₅ O₃ ;Molecular Weight of 225.21. It is an orally active acyclic nucleosidewith inhibitory activity towards several herpes viruses. The preparationof acyclovir is described in U.S. Pat. No. 4,199,574.]

The crude preparation of antiviral agent AN-1 did not inhibit eitherherpes simplex virus type 1, herpes simplex virus type 2, humancytomegalovirus or varicella zoster virus in vitro at the 100 μg/mllevel.

EXAMPLE 4 Use of A. niger Fermentation Extract as a Topical TherapeuticAgent

An excellent animal model system for the in vivo study of humanpapilloma virus related diseases can be found in rabbits using the NIH(National Institutes of Health) cottontail rabbit papillomavirus (CRPV)rabbit model system. This system has been used previously for thetesting of putative viral agents.

CRPV is naturally endemic in Midwestern cottontail rabbits, producingcutaneous papillomas in which about 25% of these lesions progress toinvasive carcinomas. Inoculating CRPV onto the skin o#domestic rabbitsconsistently produces warts. Thus, the CRPV rabbit model system can beused to test the efficacy of various antiviral agents for their abilityto prevent or mitigate wart growth.

A controlled study of the therapeutic effects of the crude cell-freefreeze-dried fermentation extract (AN-1), obtained as described above inExample 1, was performed in the NIH CRPV-rabbit model system. In thefirst stage of this study the presence and size of the tumors werecompared in animals which had either: a) received topical treatmentswith only 50% (v/v) glycerol (Group 1); or b) received 8% (w/v) AN-1 in50% (v/v) glycerol (Group 2); or c) received no treatment at all untilnine weeks following infection with CRPV and then treatment with 8%(w/v) AN-1 in 50% (v/v) glycerol. In this fashion, both the prophylacticas well as the therapeutic properties of the compositions of the presentinvention could be studied by reference to the Group 2 (for prophylacticproperties) and Group 3 (for therapeutic properties) in comparison tothe Group 1 and the control group.

METHODS AND TECHNIQUES

1. Preparation of the Virus

CRPV was isolated from the tumors of wild rabbits and prepared bystandard methods described in (2) which produced a 10% (w/v) homogenateof cottontail rabbit warts cleared of cellular debris. The virus wastitred by serial dilution and scarification on domestic female DutchBelt Rabbits, as described in (3), thereby producing warts in about 3 to4 weeks. A portion of this virus was then purified by isopycnic CsCldensity gradients, as described in (4) giving the purified virion.

The excised tumors were then frozen in liquid nitrogen, pulverized in amortar and pestle and a 10% (w/v) suspension prepared as described by(4). The viral supernatant was then applied to a velocity step gradientof CsCl at 43 g/100 ml, 32 g/100 ml and 27 g/100 ml and subjected tocentrifugation at 70,000 g (in an SW27 rotor) for two hours at 18° C.,as was described by (2). The viral band was then collected, dialyzed for48 hours, diluted and made to a density of 1.34 g/ml with CsCl as wasalso described by (2). The virus was then banded in a SW 50.1 rotor at100,000 g for 40 hours at 18° C., collected and dialyzed, as was furtherdescribed by (2).

2. Experimental Protocol

Two rabbits (C 1 and C 2) were immunized with purified CRPV virions(obtained as described above) as described in (2).

Three groups of seven rabbits each were infected with CRPV obtained asdescribed above. The back of each rabbit was infected at eight sites(four sites on the left-hand side and four sites on the right-hand side)with 50 μl of a 1:4 dilution of the stock virus (approximately 32 ID₅₀units). Such infection was performed by scarification, as described in(3).

One week later, topical treatments began. The specimens of Group 1 (thecontrol group) were given twice daily treatments with 100 μl of 50%(v/v) glycerol in deionized water. The specimens of Group 2 were giventwice daily treatments with 100 μl 8% (w/v) AN-1 in 50% (v/v) glycerolin deionized water. The specimens of Group 3 were given twice dailytreatments with 100 μl of the same composition as was given to thespecimens of Group 2, but such treatment were not started until thestart of the ninth week after infection, at which time most of theinfection sites had already developed tumors. Treatment was continuedfor two months for each group.

Treatments were effectuated by contacting the infected tumor sites withthe particular treatment and maintaining such contact for approximatelyfive (5) seconds with a "rubber policeman" to let the treatmentswork-in.

3. Isolation of Cellular DNA

Tumor tissues from each of the specimens of the three groups of section2 were extracted, minced and treated (digested) with proteinase, as isdescribed by (5). Potassium chloride and the ethanol were then added tothe cooled digest to precipitate, respectively, the protein complexestherein and the total cellular nucleic acids therein as is described by(5). RNA was then removed by treatment with ribonuclease A followed bysodium dodecyl sulfate-proteinase digestion, phenol-chloroformextractions and ethanol precipitations, as was also described by (5).

4. Preparation of Radiolabeled CRPV DNA

CRPV DNA was then obtained from the DNA isolated as described above insection 3 and molecularly cloned into pBR322 (Clontech Laboratories,U.S.A.) as described in (2). The cloned CRPV DNA was then excised fromits plasmid vector by treatment with EcoRI followed by agarose gelelectrophoresis and electroelution of the appropriate DNA band asdescribed in (2). This CRPV DNA was then radiolabeled with ³² P-dCTP bynick translation as described in (6). Specific activities of about 3×10⁸cpm/μg were customarily achieved.

5. DNA Analysis

DNA filter hybridizations were performed under stringent conditions asdescribed by (5). Cellular DNA was extracted as described in (5) fromthe DNA isolated as described above in section 3 with protease anddetergent, phenol and chloroform, and subsequently analyzed by thepolymerase chain reaction (PCR) using GeneAmp (TM) (Perkin-Elmer,U.S.A.) and oligomer primers derived from the CPRV E6 open readingframe. The sequences used for the primers were:

    5'-GAACTGCCTGCCACGCTCGC-3' SEQ ID NO:1

    5'-CGCCTGGCCCTAGGTCAAC-3'. SEQ ID NO:2

After 35 cycles, amplification of 0.5 μg of cellular DNA followed byhybridization to a radiolabeled CRPV DNA probe could detect less than 1fg of CRPV DNA in the original samples. See (3).

6. Serological Assays

Peripheral blood was drawn from each of the specimens of section 2 forenzyme-linked immunoabsorbent assay (ELISA) tests for humoral antibodyto CRPV virion proteins. Purified virions described above in section 1of this example were used in combination with Freund's complete andincomplete adjuvants to produce anti-CRPV sera as described in (7).Using long of purified virion proteins in standard ELISA assays asdescribed in (7), these sera were found to have titres of 7.7×10⁴ to4.7×10⁵. These sera served as positive controls in ELISA assays [asdescribed in (7)] for the detection of humoral CRPV antibodies inexperimental animals. Serum reactivity to the Aspergillus nigerfermentation extract (AN-1) was determined using 1 mg of the lyophilizedpowder (obtained as described above in Example 1) as the antigen. Inboth sets of assays, sera from rabbits both pre-infection and from thetermination of the study were analyzed. Comparisons in which the titreincreased by a factor of at least 4 were considered significant.

7. Statistical Analysis

The significance of the presence or absence of warts in the individualsample groups compared to the control groups was measured by achi-square test described in (8). Differences in positivity of serumantibody response were assessed using a Fisher Exact Test as describedin (9), (10) and (11). "p values" were calculated in the mannerdescribed in (9)-(11) using the Fisher Exact Test. As used herein, pvalues refer to probability values. Probability values (p) of 0.05 orless are considered significant, indicating that there is less than a 5%chance that such a result occurred randomly.

8. Results

A. Clinical Observations

The results of the clinical observations hereafter discussed aresummarized in Table 1, as follows:

                                      TABLE 1                                     __________________________________________________________________________    Clinical Observations of Effects of Topically Applied A. niger                Fermentation Extract on Growth and Removal of Warts by CRPV                                         Infection Sites W/Tumors                                           # of  # of After 56 Days                                                                          After 112 Days                                 Group                                                                             Treatment                                                                            Specimens                                                                           Sites                                                                              No.                                                                              Percent                                                                             No.                                                                              Percent                                     __________________________________________________________________________    1   Control                                                                              7     56   54 96    42 75                                          2   7 days post                                                                          7     56   45 80    24 43                                              Infection                                                                 3   56 days post                                                                         7     56   53 94    32 57                                              Infection                                                                 __________________________________________________________________________

Eight weeks after the begining of treatment, tumors were observed in 96%of the infection sites of animals in Group 1 (which received none of thecomposition) and in 94% of the animals in Group 3 (p<0.01), who had notyet received any treatment whatsoever. In comparison therewith, tumorswere observed in only 80% of the animals in Group 2. Group 2 was thegroup who had received prophylactic treatments with the composition andthe only specimens who, until this time, had received any of thecomposition. Thus, it can be seen that when applied topically for anextended period begining within one week of infection by CRPV, there wasa 17% reduction in the number of tumors observed when compared tocontrols after eight weeks. An additional nine weeks later (withoutfurther treatment) the specimens demonstrated a 43% reduction in thenumber of tumors observed when compared to controls.

These 17% and 43% reductions in tumors demonstrate the prophylacticproperties of the composition of the present invention.

Relative to the specimens of Group 3 (the Group who had received thetherapeutic treatments with the composition), it was found that whenAN-1 was repeatedly topically applied to specimens having existingtumors (present nine weeks after CRPV infection), after about eightweeks, such specimens experienced 24% fewer tumors than controlspecimens (the percentage of infection sites with tumors for specimensof Group 3 was 57% with a p>0.05). This 24% reduction in tumorsdemonstrates the therapeutic properties of the composition of thepresent invention.

In summary then, this study demonstrates both the prophylactic andtherapeutic properties of the compositions of the present invention forthe prevention and treatment of viral-induced tumors.

1) Skin Reactions

It is noted here that several of the specimens in each Group developed"rashes" (red, lumpy areas) after a few weeks of AN-1 administrationwhich were initially only in the vicinity of the drug administration.However, as the experiment progressed, we found rashes to be more widelyspread.

The skin rashes observed are reminiscent of red skin and itchiness whichis observed in regressing warts in patients.

It is suspected that the specimens were experiencing delayed allergicresponses as a result of previous or extended exposure to Aspergillusniger antigens. Delayed hypersensitivity, such as was observed, is acellular immune response. Necropsy of animals revealed no untoward sideeffects on major organs. In some of the tumor sites on AN-1 treatedanimals, superficial dermas were infiltrated with moderate numbers oflymphocytes and small numbers of macrophages and plasma cells. Similarinfiltrates were found in the non-papillomavirus nodules associated withthe hypersensitivity response.

2) Wart Regression

112 days after infection, all groups showed some tumor regression. Atthis time, the percentage of infection sites with tumors for Group 1 was75%. Therefor, there was a 25% tumors regression. There was anadditional 24% wart regression in tumors in the specimens of Group 3 dueto treatment with the fermentation extract. However, some of the tumorsin Group 3 which had regressed were tumors which were in the process ofregression prior to treatment with AN-1, as determined by intermediatetumor size measurements.

B. Serological Observations

Serological studies testing reactivity to CRPV and AN-1 antigens byELISA assays of both pre-study sera and post-study sera from allspecimens is shown in Table 2. C 1 and C 2 stand for, respectively,Control Specimen #1 and Control Specimen #2, which served as controlsfor this ELISA assay:

                                      TABLE 2                                     __________________________________________________________________________    ELISA Assay Results Using CRPV and AN-1 Antigens                              Titre of Sera From Each Animal                                                    Pre-Sera                                                                           Post-Sera                                                                          Ratio                                                                              Pre-Sera                                                                            Post-Sera                                                                           Ratio                                                                              # of                                      Spec.                                                                             AN-1 Ag                                                                            AN-1 Ag                                                                            Post/Pre                                                                           CRPV Ag                                                                             CRPV Ag                                                                             Post/Pre                                                                           Tumors                                    __________________________________________________________________________    Group 1 (Control Group)                                                        1  160  160  1    320   320   1    8                                          2  10   10   1    320   40    ˜1                                                                           8                                          3  10   40   4    10    40    4    7                                          4  20   40   2    160   320   2    8                                          5  20   20   1    20    40    2    0                                          6  10   10   1    10    10    1    8                                          7  20   20   1    10    20    2    3                                         Group 2                                                                        8  10   20   2    10    10    1    8                                          9  10   10   1    10    10    1    8                                         10  10   10   1    10    10    1    0                                         11  20   160  8    20    160   8    0                                         12  10   10   1    10    10    1    0                                         13  10   20   2    10    20    2    8                                         14  40   40   1    40    40    1    0                                         Group 3                                                                       15  160  640  4    160   640   4    3                                         16  320  320  1    320   160   ˜1                                                                           8                                         17  80   1280 16   80    640   8    2                                         18  40   40   1    40    160   4    8                                         19  40   40   1    20    20    1    0                                         20  10   40   4    10    80    8    8                                         21  160  640  4    20    160   8    3                                         C 1 320  360  ˜1                                                                           320   77760 243  Immunized                                 C 2 2560 2160 ˜1                                                                           640   466560                                                                              729  Immunized                                 __________________________________________________________________________

Sera were diluted 1:10 and then two-fold dilutions thereafter. Positivecontrol sera was diluted 1:10 and six-fold dilutions therelater. Titreis inverse of dilution. End points are the last dilution with anAbsorbance at 490 nanometers of 0.1 or greater. Increases of four-foldor more are considered significant. Controls consisted of specimenswhich had been immunized with the purified CRPV virions as describedabove (3).

The results seen above relative to Table 2 show that five (5) animals inGroup 3 had a significant sero-positive response (a ratio of post to preof four or more) to CRPV antigens, as opposed to only one (1) specimenin Group 1. This difference in the responses to CRPV antigens betweenthe specimens of Group 1 (which received none of the composition of thepresent invention) and the specimens of Group 3 (which received atherapeutic treatment of the composition of the present invention) wassignificant by the Fisher Exact Test (p=0.05).

It is also noted that four specimens in Group 3 had a seropositiveresponse to AN-1 antigen, as compared with only one specimen in Group 1.While this is felt to be further indicative of the efficacy of thecomposition of the present invention, statistically, such a ratio is notsignificantly different from the response in Group 1 control animals.

While it is noted that the elevated antigen responses did not correlatewith the observed tumor growth or lack thereof, generally an increase inresponse to one antigen paralleled an increase in response to the otherin Groups 2 and 3. The role of this serological response to wartregression is not clear. These results would suggest a generalizedhypersensitivity, as observed by the skin reactions, as the pathway bywhich AN-1 enhanced regression of papillomas.

C. Molecular Observations

PCR-hybridizations analyses as described in (3) were performed of thetumor negative sites in the specimens of the three groups in the currentstudy to detect the presence of CRPV in virtually every CRPV-inducedtumor. The investigation showed the presence of CRFV DNA in as many as23% (3 of 13) of the tested sites in Group 1 as compared to only 6.5% (2of 31) of the tested sites in Group 2 and 9.5% (2 of 21) of the testedsites in Group 3 (see Table 3). In contrast, CRPV DNA was found in 76%of a sampling of tumors used as positive controls.

                                      TABLE 3                                     __________________________________________________________________________    Results of PCR DNA Analyses                                                   Group 1         Group 2     Group 3                                           Tumor.sup.1 :                                                                       +    -    +     -     +     -                                           Total 4/5  3/13 2/4   2/31  7/8   2/21                                        Positive.sup.2                                                                      (80%)                                                                              (23%)                                                                              (50%) (6.5%)                                                                              (88%) (9.5%)                                      __________________________________________________________________________    Results.sup.3                                                                       1:01 5:01  8:01 10:01 15:02 +                                                                             15:05                                             3:08 +                                                                             5:02  8:02 +                                                                             10:02 16:01 +                                                                             15:06                                             7:01 +                                                                             5:03  9:01 +                                                                             10:03 17:01 +                                                                             15:07 +                                           7:02 +                                                                             5:04 +                                                                             13:01 10:04 +                                                                             17:08 +                                                                             15:08 +                                           7:03 +                                                                             5:05 +     10:05 18:01 +                                                                             17:03                                                  5:06       10:06 18:04 +                                                                             17:05                                                  5:07       10:07 +                                                                             20:01 +                                                                             17:06                                                  5:08       10:08 21:06 17:07                                                  7:04       11:01       19:01                                                  7:05 +     11:02       19:02                                                  7:06       11:03       19:03                                                  7:07       11:04       19:04                                                  7:08       11:05       19:05                                                             11:06       19:06                                                             11:07       19:07                                                             11:08       19:08                                                             12:01       21:01                                                             12:02       21:02                                                             12:03       21:04                                                             12:04       21:05                                                             12:05                                                                         12:06                                                                         12:07                                                                         14:01                                                                         14:02                                                                         14:03                                                                         14:04                                                                         14:05                                                                         14:06                                                                         14:07                                                                         14:08                                                   __________________________________________________________________________     .sup.1 Infection sites were extracted for total DNA, amplified by PCR for     the presence of CRPV DNA and analyzed by Southern Blot Analysis as            described by (3). All tumor negative sites were examined in each of the       three study groups. Only a representative sampling of tumor positive site     were examined from each group.                                                .sup.2 Number CRPV DNA positive over number tested (%). This is a summary     of the raw data presented in this Table.                                      .sup.3 Raw Data as follows: Rabbit Number:Infection Site CRPV DNA             Positivity.                                                              

These results in the rabbit model system would seem to indicate theefficacy of the composition for prophylactic and therapeutic purposes inhumans as well as other mammmals.

EXAMPLE 5

Use of A. niger Fermentation Extract as a Topical Therapeutic Agent

Two horses, each with wart-type tumors on their noses were treated for atwo week period. The liquid filtered fermentation extract composition,obtained as described above in Example 1 (but before the freeze-dryingthereof), was topically applied with a cotton gauze to the warts on oneside of each of the horse's noses. As a control, sterile phosphatebuffered saline (PBS) was topically applied with a cotton gauze to thewarts on the other opposite side of each of the horse's noses.

1. Protocol

Treatments were conducted by placing 30 ml of the solutions (thecomposition of the PBS controls) onto several layers of gauze anddabbing it onto the warts until soaked. This was repeated twice duringeach treatment, so that the afflicted area was soaked. Treatments weremade on days 0, 1, 2, 3, 7 and 14. Observations were made on days 0, 1,2, 3, 7, 14, 21 and 30.

2. Results

By approximately 7 days, post-initiation of treatments of warts on thehalf of the muzzle being treated with the composition began changing inappearance (they appeared whiter, whereas others had a pink appearance).

By day 21, it was noted that the warts on the PBS half were much largerand it appeared as if they had grown in numbers. These warts appearedpink and quite viable. The warts on the composition-treated side werestill present, however, they did not appear to have grown in either sizeor number. Again, they appeared white and quite crusty.

By 30 days, there appeared to be no change from 21 days.

3. Conclusions

The composition of the present invention exhibited a therapeutic effecton warts. The PBS-treated warts grew larger and spread, indicating thatthe warts were young at initiation of both treatments. Therefor, thedifference in appearance between PBS-treated and the composition treatedwarts at 30 days is significant.

EXAMPLE 6

Use of A. niger Fermentation Extract as a Topical Therapeutic Agent.

Another excellent animal model system for the in vivo study of humanpapilloma virus related diseases are severe combined immunodeficient(SCID) mice.

SCID mice are chimeric animals in which a mouse has been engrafted withcells, from another animal. These SCID mice show little evidence ofgraft-versus-host disease. Because the introduced cells of the engraftedSCID mouse support infection, SCID mice are an attractive model for thetesting of therapeutic and prophylatic compositions. A full descriptionof SCID mice can be found by reference to Milman, G., and D'Souza, P.,ASM News (1990) Vol. 56, No. 12 at 639-642, the contents of which areincorporated herein.

This model involves infecting, with cottontail rabbit papilloma virus(CRPV), New Zealand White (NZW) rabbit ear skin that has beentransplanted to the dorsum of a SCID mouse. The potential anti-PVtherapy was then evaluated for its ability to inhibit wart growth. Sincethe SCID mouse is immunologically deficient in T and B cell function,this model examines the efficacy of the therapeutic and prophylacticcompound of the present invention (the A. niger fermentation extractAN-1) without regard to any input from these immunological factors.

A controlled study of the therapeutic effects of the freeze-dried crudecell-free fermentation extract (AN-1), obtained as described above inExample 1, was performed as follows:

Materials and Methods

CRPV stocks:

Viral stocks were prepared by grinding cottontail rabbit warts toproduce a 10% (w/v) homogenate which was then centrifuged to remove thecellular debris. Supernatants were stored at -70° C. until use.

Transplant and infection of NZW skin:

NZW rabbit ear skin was transplanted to both the left and right dorsumof anesthetized mice and allowed to heal for several weeks. The tissueon both flanks was then infected by scratching with a 27 G needle (100×)and placing a 5 μL droplet of undilute CRPV inoculum on the abradedtissue.

Preparation of Compounds:

All the compounds were prepared in an aqueous cream to a finalconcentration of 8% (w/v) of the freeze-dried extract with 10% (v/v)dimethyl sulfoxide (DMSO) added. 400 mg of compound was added to 4.1 gof cream and stirred for 15 minutes. To aid in dissolution the compoundswere placed in a 56° C. water bath for 1 hour. Finally, 0.5 ml of DMSOwas added and the compounds were again stirred. Compounds were stored at4° C. between treatments.

Treatment of Lesions:

Topical therapies were applied to both the left and right CRPV infectedgrafts twice a day starting on day 3 post infection (PI) and continuedthrough week 6 post infection (PI). There were 5 mice/treatment groupfor a total of 10 infected grafts/group.

Evaluation of Efficacy:

Treatment efficacy was evaluated weekly from week 2 through week 8 usingthe following lesion scoring system:

    ______________________________________                                        SCORE        CLINICAL DESCRIPTION                                             ______________________________________                                        0            No infection visible                                             1            Thickening of skin at infection site                             2            Small, discrete papillomas                                       3            Large, discrete papillomas                                       4            Semi-confluent papillomas                                        5            Confluent papillomas                                             6            Dense keratin horn                                               ______________________________________                                    

For data analysis, lesion scores of the grafts in a treatment group wereaveraged together. Areas under the curves (AUC) where determined and thepercent reduction in AUC for each treatment was calculated with respectto untreated control animals.

Results:

The results are summarized below in Table 4:

    ______________________________________                                        TREATMENT                                                                     WEEK POST                    TREATMENT                                        INFECTION    NO TREATMENT    WITH AN-1                                        ______________________________________                                        1            0.0             0.0                                              2            0.5             0.7                                              3            1.2             1.0                                              4            2.3             1.7                                              5            4.4             3.2                                              6            5.4             4.1                                              7            6.0             4.8                                              8            6.0             5.0                                              AUC          22.8            18                                               % REDUCTION                  21.1                                             ______________________________________                                    

In this model, a reduction in AUC of greater than 20% is consideredsignificantly different from untreated animals and may be indicative ofan effective treatment. Use of the crude, cell-free A. nigerfermentation extract (AN-1) produced a reduction in AUC of 21.1%. Thus,it can be seen that use of the AN-1 fermentation extract produced asignificant reduction in wart development when tested in this model.

Many modifications may be made without departing from the basic spiritof the present invention. Accordingly, it will be appreciated by thoseskilled in the art that within the scope of the appended claims, theinvention may be practiced other than has been specifically describedherein.

Bibliography

(1) Takeshima, H., Antiviral Agents. In The Search for BioactiveCompounds from Microorganisms, Omura, S., ed., Springer-Verlag, N.Y.,N.Y. (1992) at page 50.

(2) Watts, S., et al., (1983) Virology 125 at 127-138.

(3) Ostrow, R., et al., (1992) Antiviral Res. 17 at 99-113.

(4) Ostrow, R., et al., (1982) Proc. Natl. Acad. Sci. USA 79 at1634-1638.

(5) Manias, D., et al., (1989) Cancer Res. 49 at 2514-2519.

(6) Ostrow, R., et al., (1981) Virology, 108 at 21-27.

(7) Poindexter, N., and Schlievert, P., (1985) J. of Infect. Dis. 151 at65-72.

(8) Dowdy, S., and Wearden, S., Chi-Square Distributions In: Statisticsfor Research, J. Wiley & Sons, Inc., N.Y., N.Y. (1983) at 97-124.

(9) Latscha, R., (1955) Biometrika 40 at 74-86.

(10) Finney, D. J., (1948) Biometrika 35 at 145-156.

(11) Fisher, R. A., Statistical Methods for Research Workers, 14th ed.,Hafner, N.Y., N.Y. (1973).

(12) Lidin, B., et al., (1992) Antiviral Res. 17 at 79-89.

(13) Korba, B. E. and J. L. Gerin (1992) Antiviral Res. 19, 55-70.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acids : synthetic                           oligonucleotide                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GAACTGCCTGCCACGCTCGC20                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acids : synthetic                           oligonucleotide                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CGCCTGGCCCTAGGTCAAC19                                                         __________________________________________________________________________

What is claimed is:
 1. A prophylactic and therapeutic composition forthe prevention and treatment of viral-induced tumors in mammals,comprising an Aspergillus fermentation extract or a derivative thereofin a pharmaceutically-acceptable carrier, wherein said Aspergillusfermentation extract is obtained from an Aspergillus niger strainselected from the group consisting of NRRL Accession No. 21139 and NRRLAccession No.
 21126. 2. The prophylactic and therapeutic composition ofclaim 1, wherein said Aspergillus fermentation extract is water-soluble.3. The prophylactic and therapeutic composition of claim 1, wherein saidviral-induced tumors comprise pappillomavirus-induced tumors.
 4. Afermentation extract of NRRL Accession No. 21139, and derivativesthereof.
 5. A fermentation extract of NRRL Accession No. 21126, andderivations thereof.